Gene regulation of retinal cell differentiation in development is crucial for the generation of mature and functional retinal neurons that form the cellular basis of visual function. Dysregulation in retinal development causes developmental abnormalities of eye and impairs visual function. Stem cell-based regenerative medicine and cell replacement therapy require a better understanding of key developmental regulators and signal pathways in retinal cell differentiation. Our long-term goal of this proposal is to elucidat those molecular mechanisms that control the maintenance of multipotential retinal progenitor cells (RPCs) and their subsequent photoreceptor cell commitment. We predict that RPC-multipotency factors (providing differentiation potentials) and lineage-specific signals act in concert to initiate the expression of decision-making gene(s) that steer(s) a subset of RPCs towards the photoreceptor lineage. However, not much is known about the molecular events in the regulation of RPC multipotency and the initiation of photoreceptor cell differentiation. Homeodomain transcription factors Six3 and Six6 are structurally similar and largely co-expressed in multipotent RPCs in mouse. Our current preliminary studies have identified severe RPC defects in Six3; Six6 compound null retinas; these RPC defects are not evident in either Six3 or Six6 single null retinas. In photoreceptor cell differentiation, homeodomain transcription factor Otx2 is the earliest marker and the essential upstream regulator in a gene network that direct photoreceptor precursors to either rods or cones. Thus, Otx2 is a decision-making gene whose expression marks cell commitment, and dissection of the regulatory components for its neuroretinal expression help unravel as yet unknown molecular mechanisms that promote RPCs to differentiate into photoreceptor precursors. Now we have identified a novel, highly conserved enhancer that recapitulates the initial Otx2 retinal expression. In this proposal we aim 1) to elucidate the regulatory pathways under the control of Six3/Six6 joint functions in retinal cell differentiation and 2) to determine the molecular mechanisms that control Otx2 retinal expression through this Otx2 retinal enhancer. The outcomes of these studies will provide insights into the molecular mechanisms controlling the maintenance of multipotential retinal progenitor cells and photoreceptor cell differentiation, and thus facilitate the design of novel strategies for the generation of specific retinal cells, e.g., photoreceptor cells, from the culturs of human embryonic stem cells or induced pluripotent stem cells in the endeavor of modeling and treatment of human retinal diseases.